Health and environmental related fields face various biochemical processes, which have to be evaluated rapidly at decreasing detection levels. A lot of biochemical analytical methods involve immobilisation of a biological sensing element on a surface. The increasing miniaturisation of the detection system and the demand for a high sensitivity impose more severe demand on the immobilisation of such biological sensing elements. Generally, a biosensor comprises a biological sensing element, such as, e.g., an antibody or single-stranded DNA, in close contact with a physico-chemical transducer, such as e.g., an electrode. Measurement of a target molecule, e.g., an antigen or a complementary DNA strand, in an analyte is achieved by selective transduction of a parameter of the biomolecule-analyte interaction into a quantifiable signal.
Two important and desired properties of a biosensor are its specificity and its sensitivity towards the target molecule(s). Biosensors must also fulfil major requirements like stability, speed and reproducibility. In addition, the analyte must be detectable in an excess of other proteins. Because of these strict requirements, affinity biosensors are not yet commercially available. More specifically, the intrinsically high specificity of biomolecular systems can be successfully exploited for the realisation of highly sensitive integrated biosensor devices only if a highly efficient coupling between the biological and transducer components is realised [M. P. Byfield and R. A. Abuknesha, in ‘Biochemical aspects of biosensors’, Biosensors and Bioelectronics, 9 (1994) pp. 373400]. Therefore, the biological sensing elements are preferably bound on the surface in such a way that a significant and specific interaction with the target molecules occurs. Moreover, a well-defined interface between the transducer surface and the biomolecules will allow control over the reproducibility of the biosensor device and over the extent of non-specific adsorption of any undesired biospecies. For immuno-sensors the most common biological sensing elements are antibodies and specific binding proteins, which have a reversible specific binding affinity for an analyte.
DNA chip technology uses microscopic arrays of DNA molecules immobilized on solid supports for biomedical analysis such as gene expression analysis, polymorphism or mutation detection, DNA sequencing and gene discovery [G. Ramsay, ‘DNA chips: state-of-the art’. Nature Biotechnol., 16 (1998) pp. 40-44]. DNA chips generally comprise a glass substrate, as glass is a popular material for DNA chip technology due to its low fluorescence, transparency, low cost and resistance to high temperatures. A variety of non-covalent coupling chemistries have been used to bind DNA onto glass, such as, for example, polylysine coatings or hydrophobic interactions. However, in these cases, DNA films are susceptible to removal from the surface under high salt or high temperature conditions. Covalent binding methods are thus preferred. Usually, DNA is cross-linked by ultraviolet irradiation to form covalent bonds between thymidine residues in the DNA and positively charged amino groups added on the functionalised slides. Alternatively, DNA molecules are fixed at their extremities. Thus, carboxylated or phosphorylated DNA can be coupled to aminated supports as well as the reciprocal situation [S. S. Ghosh and C. F. Musso, ‘Covalent attachment of oligonucleotides to solid supports’. Nucleic Acids Res., 15 (1987) pp. 5353-5372]. Amino-terminal oligonucleotides can also be bound to isothiocyanate-activated glass, or to glass surfaces modified with epoxide. Thiol-modified or disulfide-modified oligonucleotides have also been grafted onto aminosilane via a heterobifunctional crosslinker or on 3-mercaptopropylsilane. Zammateo et al. reported that the coupling of aminated DNA to glass supports derivatised with aldehyde groups presents several advantages over other methods [N. Zammatteo et al., ‘Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays’. Anal. Biochem., 280 (2000) pp. 143-150]. One of them is that aminated DNA is directly bound without the help of a coupling agent. Time-consuming reactions and unstable reagents such as coupling activators are avoided, making this procedure well suited for DNA chip technology. One drawback is that the aldehyde-amine condensation leads to the formation of imine groups, which are not very stable and must be reduced in stable amines by borohydride treatment.
To date, the main method for the immobilisation of biological moieties on oxide and glass surfaces has involved reaction with functional organo-alkoxysilanes, followed by a covalent attachment of the biological molecule to the newly introduced functional groups on the surface. Examples of silane molecules frequently used for this purpose are (3-aminopropyl)triethoxysilane (APTES), (3-mercaptopropyl)trimethoxysilane (MPTMS) and (3-glycidoxypropyl)dimethylethoxysilane (GPMES). Glass or oxide surfaces can also be modified by silane chemistry to introduce aldehyde functions [N. Zammatteo et al., ‘Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays’. Anal. Biochem., 280 (2000) pp. 143-150]. Because this method is a relatively simple means to introduce a variety of functional groups on oxide materials and glass, it is frequently used in the realisation of affinity biosensors. Nevertheless, it is known that the use of these short-chain trialkoxysilanes often leads to highly polymeric, less effective and heterogeneous surfaces, which is a potential disadvantage when preparing surfaces for biosensors [K. Bierbaum, et al., ‘A near edge X-ray absorption fine structure spectroscopy and X-ray photoelectron spectroscopy study of the film properties of self-assembled monolayers of organosilanes on oxidised Si(100)’. Langmuir, 11 (1995) pp. 512-518]. These drawbacks can be ascribed primarily to the short alkyl chain length of these molecules (typically propyl-), which is unable to lead to energetically favourable intermolecular (i.e., Van der Waals) interactions, to the relatively low reactivity of the silicon—(m)ethoxy bond and to cross-reactivity of the functional groups (e.g., amino) with the oxide surface.
In order to create a well-defined interface between the transducer surface and the biomolecules, the silane molecules preferably assemble on the surface of a substrate in a uniform monolayer. In theory, this kind of arrangement allows the biomolecules to be attached to the functional moiety of the silane in a similarly uniform fashion. Such a well-organised interface would be of great value for a higher sensitivity and reproducibility of biosensors.
As opposed to the commercial alkyltriethoxysilanes with short alkanes, most commercial alkyltrichlorosilanes generate well-structured self-assembled monolayers (SAMs), when produced under the proper conditions [J. D. Brzoska et al., ‘Silanization of solid surfaces: a step toward reproducibility’. Langmuir 10 (1994) pp. 4367-4373]. The SAM formation from chlorosilanes is mainly induced by the longer alkyl chains of the available chlorosilanes (3 to 18 CH2's), compared to most available ethoxysilanes that only comprise 3 CH2's. These long alkyl chains enable energetically more favourable intermolecular (i.e., Van der Waals) interactions, resulting in the possible spontaneous formation of ordered monolayers. The use of SAMs provides an easy way to functionalise surfaces by formation of a highly ordered uni-molecular film, with the flexibility to design different head groups on the monolayers. More specifically, hydrophobic and hydrophilic groups at the end of the monolayers provide an excellent possibility to immobilise enzymes, proteins, or whole cells for selective sensing of different analytes. Especially for alkanethiols on metal surfaces, this versatile SAM formation is already a reality [J. C. Love, ‘Self-Assembled Monolayers of Thiolates on Metals as a Form of Nanotechnology’, Chemical Reviews, ASAP article, 2005].
Unfortunately however, many polar groups suitable for biomolecule immobilisation (such as COOH, NH2 and SH) are incompatible with the highly reactive (i.e., electrophilic) trichlorosilane group. Therefore, such polar functional groups are usually generated from non-polar precursors such as, e.g., Br, C═C, C≡N and others, after formation of a monolayer film of the latter molecules on the biosensor surface [S. R. Wasserman et al., ‘Structure and reactivity of alkylsiloxane monolayers formed by reaction of alkyltrichlorosilanes on silicon substrates’. Langmuir 5 (1989) pp. 1074-1087]. To date, the requirement for a post-silanisation surface reaction and the stringent conditions necessary to allow for monolayer formation are the major drawbacks for the implementation of alkylchlorosilanes in the process of immobilising biomolecules, which explains their rare exploitation in biosensor development and micro-array applications.
To allow for surface modification after silanisation, non-nucleophilic terminal groups have to be introduced on the surface first. To this end, SAMs from alkyltrichlorosilanes with termini such as halogen, cyanide, thiocyanide, ether, ester, thioether, thioester, vinyl, α-haloacetate and p-chloromethylphenyl have already been deposited [A. Ulman, ‘Formation and structure of self-assembled monolayers’. Chem. Rev., 96 (1996) pp. 1533-1554]. Some of the afore mentioned terminal groups can also be created in situ, for example, by nucleophilic substitution on bromine terminated SAMs, giving rise to thiocyanato, cyanide and azide surfaces. Afterwards, they can be converted to the corresponding mercapto and amino terminated monolayers, by reduction with LiAlH4. Also the modification of vinyl groups by oxidation to obtain hydroxyl or carboxyl terminated monolayers has been studied [L. Netzer and J. Sagiv, ‘Adsorbed monolayers versus Langmuir-Blodgett monolayers—why and how?: From monolayer to multilayer, by adsorption’. J. Am. Chem. Soc., 105 (1983), pp. 674-676]. Lee et al. have extensively investigated the reactivity of various halogen terminated SAMS towards nucleophiles such as amino and thiol compounds [Y. W. Lee at al., ‘Electrophilic siloxane-based self-assembled monolayers for thiol-mediated anchoring of peptides and proteins’. Langmuir, 9 (1993) pp. 3009-3014]. It was found that the reactivities of the functionalised monolayer were in the order: α-haloacetyl>benzyl halide>>alkyl halide. Moreover, within each class of compounds the reactivity follows the order of leaving groups: I>Br>Cl directly. The most reactive compound, i.e., α-iodoacetyl, has been used to bind cysteine-containing peptides. Also Fryxell et al. have studied nucleophilic displacements between halide terminated SAMs and anionic nucleophiles or between ester terminated SAMs and neutral nucleophiles [G. E. Fryxell et al., ‘Nucleophilic displacements in mixed self-assembled monolayers’. Langmuir 12 (1996) pp. 5064-5075]. These synthetic elaborations were also carried out on mixed monolayers in order to create SAMs with a mixed functionality. An alternative approach for the realisation of functional surfaces from alkyltrichlorosilane SAMs is the deposition of protected sulphur containing or (thio)ester terminated alkyltrichlorosilanes. After deprotection, thiol, or carboxyl terminated SAMs can be created [S. R. Wasserman et al., ‘Monolayers of 11-trichlorosilylundecyl thioacetate: a system that promotes adhesion between silicon dioxide and evaporated gold’. J. Mater. Res., 4 (1989) pp. 886-892]. However, these protected precursor-silanes often require complex synthesis and/or harsh de-protection conditions. The two most frequently applied post-silanisation surface reactions are the oxidation of vinyl terminated SAMs to create carboxyl groups [J. H. Schon and Z. Bao, ‘Organic insulator/semiconductor heterostructure monolayer transistors’. Appl. Phys. Lett., 80 (2) (2002) pp. 332-333] and the nucleophilic substitution of bromine terminated SAMs with azide, followed by reduction, to create amino functional surfaces. These surface modifications have been used in applications ranging from fundamental studies of surface reactions, over controlled multilayer formation and fabrication of organic transistors, to the immobilisation of biomolecules for biosensors.
The deposition of functional organoalkoxysilanes, as well as the performance of surface reactions on SAMs of precursor organohalosilanes, usually give rise to a functional group that has to be activated in situ before attachment of the biological moiety. Possibilities for this type of functional group include, but are not limited to, simple moieties such as carboxyl, amino or a thiol group. However, a preferred route for the immobilisation of biological moieties onto oxide and glass surfaces is the deposition of silane molecules with a highly reactive functional moiety that is compatible with monolayer formation and needs no in situ activation prior to reaction with the biological moiety. For example, the deposition and use of trialkoxysilanes with a maleimide function at the end of the alkane chain, which serves for the direct immobilisation of thiol-terminating molecules, was reported by Lu et al. [H. B. Lu et al., ‘Engineered biointerfaces for protein biochip applications’. Presented at the AVS 49th International Symposium, Denver, Colo., USA (Nov. 4-8, 2002), BI+HS+SS−ThM11]. Lee et al. have used various halogen terminated SAMs, which are reactive towards nucleophiles such as amino and thiol compounds. For SAMs of alkane thiols on gold, N-hydroxysuccinimide and maleimide terminated surfaces have been reported for the direct immobilisation of proteins.
Besides the introduction of functional groups that allow covalent binding or physical adsorption of receptor molecules, which in turn allows affinity binding with the analyte, protein-resistant functional groups can also be introduced onto the surface using SAMs. The use of oligo(ethylene glycol)- or PEG-terminated alkyltrichlorosilanes, [Cl3Si(CH2)11(OCH2CH2)nOCH3], with n=2 or 3, for the formation of protein-resistant coatings on glass and metal oxide surfaces was described by S. -W. Lee and P. E. Laibinis in ‘Protein-resistant coatings for glass and metal oxide surfaces derived from oligo(ethylene glycol)-terminated alkyltrichlorosilanes’. Biomaterials, 19 (1998) pp. 1669-1675. Also for SAMs of alkane thiols on gold, the incorporation of oligo(ethylene glycol) functionalities at the terminus has been applied for lowering the non-specific adsorption of proteins on surfaces. Functional groups, other than ethylene glycol, that give surfaces the ability to resist the non-specific adsorption of proteins from solution, have been described by E. Ostuni et al. in ‘A survey of structure-property relationships of surfaces that resist the adsorption of proteins’. Langmuir, 17 (2001), pp. 5605-5620′ and by R. S. Kane et al. in ‘Kosmotropes form the basis of protein-resistant surfaces’. Langmuir, 19 (2003), pp. 2388-2391”. However, the incorporation of these alternative groups into single-component SAMs resulted in surfaces that are comparable to, but slightly less good than, single-component SAMs that present oligo(ethylene glycol) in their ability to resist the adsorption of proteins.
Ideally, not all molecules in a monolayer are identical and mixed monolayers are created. For instance, the first molecule in the monolayer serves to immobilise a biological moiety and the second molecule contains a functional group that is resistant to the non-specific binding of biomolecules. For alkyltrichlorosilanes, mixed monolayers have been used to tailor the surface properties of a silanised surface by introducing various functional groups on the terminus of the silane monomers. It has been shown that through the use of such mixed monolayers the properties of the surface can be varied in a continuous manner from one type to another [G. E. Fryxell et al., ‘Nucleophilic displacements in mixed self-assembled monolayers’. Langmuir 12 (1996) pp. 5064-5075].
Ideally, silanes with a hydrophilic termination are preferably chosen for the creation of biosensor surfaces, because they are able to resist the non-specific adsorption of biomaterial in a further stage. Unfortunately however, such mixed silane films have not been realised yet. Frederix et al. report mixed self-assembled monolayers (SAMs) of alkane thiols on gold, in which the two functionalities are simultaneously incorporated on the SAM coated gold surface [F. Frederix et al., ‘Enhanced performance of a biological recognition layer based on mixed self-assembled monolayers of thiols on gold’. Langmuir, 19 (2003), pp. 4351-4357]. The properties of the mixed SAMs fulfill the necessary requirements to achieve stable and well-ordered mixed SAMs. Their use as an affinity biosensor interface for immuno-sensor applications was evaluated using SPR. In this study, the amount of the different components in the mixed SAMs was optimised with regard to antibody immobilization, antigen recognition, and non-specific adsorption. It was found that a mixed SAM deposited from a mixed solution of 5% of 16-mercaptohexanoic acid and 95% of 11-mercaptoundecanol exhibits the most favourable properties. Their stability and their excellent qualities concerning non-specific adsorption makes these mixed SAMs very useful as a basis for the development of affinity biosensor interfaces for real diagnostic applications or immuno-sensors.
Alternatively, oligo(ethylene glycol) functionalities are incorporated into a homogeneous protein-binding monolayer film such as, for example, self-assembled monolayers of carboxy-functionalized poly(ethylene glycol)alkane thiols [HOOC—CH2—(OCH2CH2)n—O—(CH2)11)—SH], with n=22-45, which hav protein resistant and specific antigen binding capability after covalent antibody binding onto the monolayer.
Ideally, mixed SAMs are created from molecules with a highly reactive functional moiety that needs no in situ activation prior to reaction with the biological moiety (also called pre-activated biological moieties) and from molecules that have the ability to resist the non-specific adsorption of proteins. Even more ideally, protein-resistant functionalities are also incorporated in the first type of molecule. For example, Houseman et al. have reported on such a thiol monolayer that presents both maleimide and penta(ethylene glycol) on the surface and that are able to simultaneously react with thiol-terminated ligands and prevent the non-specific adsorption of proteins onto the mixed functional surface [B. T. Houseman et al, ‘Maleimide-functionalized self-assembled monolayers for the preparation of peptide and carbohydrate biochips’, Langmuir, 19 (2003), pp. 1522-1531].
However, as these molecules comprise thiol-functional groups, they can not be attached to glass and oxide-substrates, and hence are limited in their applicability.